Bambam: parallel comparative analysis of high-throughput sequencing data

ABSTRACT

The present invention relates to methods for evaluating and/or predicting the outcome of a clinical condition, such as cancer, metastasis, AIDS, autism, Alzheimer&#39;s, and/or Parkinson&#39;s disorder. The methods can also be used to monitor and track changes in a patient&#39;s DNA and/or RNA during and following a clinical treatment regime. The methods may also be used to evaluate protein and/or metabolite levels that correlate with such clinical conditions. The methods are also of use to ascertain the probability outcome for a patient&#39;s particular prognosis.

BACKGROUND

A central premise in modern cancer treatment is that patient diagnosis,prognosis, risk assessment, and treatment response prediction can beimproved by stratification of cancers based on genomic, transcriptionaland epigenomic characteristics of the tumor alongside relevant clinicalinformation gathered at the time of diagnosis (for example, patienthistory, tumor histology and stage) as well as subsequent clinicalfollow-up data (for example, treatment regimens and disease recurrenceevents).

With the release of multiple tumor and matched normal whole genomesequences from projects like The Cancer Genome Atlas (TCGA), there isgreat need for computationally efficient tools that can extract as muchgenomic information as possible from these enormous datasets (TCGA,2008). Considering that a single patient's whole genome sequence at highcoverage (>30×) can be hundreds of gigabytes in compressed form, ananalysis comparing a pair of these large datasets is slow and difficultto manage, but absolutely necessary in order to discover the manygenomic changes that occurred in each patient's tumor.

Breast cancer is clinically and genomically heterogeneous and iscomposed of several pathologically and molecularly distinct subtypes.Patient responses to conventional and targeted therapeutics differ amongsubtypes motivating the development of marker guided therapeuticstrategies. Collections of breast cancer cell lines mirror many of themolecular subtypes and pathways found in tumors, suggesting thattreatment of cell lines with candidate therapeutic compounds can guideidentification of associations between molecular subtypes, pathways anddrug response. In a test of 77 therapeutic compounds, nearly all drugsshow differential responses across these cell lines and approximatelyhalf show subtype-, pathway and/or genomic aberration-specificresponses. These observations suggest mechanisms of response andresistance that may inform clinical drug deployment as well as effortsto combine drugs effectively.

There is currently a need to provide methods that can be used incharacterization, diagnosis, treatment, and determining outcome ofdiseases and disorders.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides methods for generating databases that may be usedto determine an individual's risk, in particular, for example, but notlimited to, risk of the individual's predisposition to a disease,disorder, or condition; risk at the individual's place of work, abode,at school, or the like; risk of an individual's exposure to toxins,carcinogens, mutagens, and the like, and risk of an individual's dietaryhabits. In addition, the invention provides methods that may be used foridentifying a particular individual, animal, plant, or microorganism.

In one embodiment, the invention provides a method of deriving adifferential genetic sequence object, the method comprising: providingaccess to a genetic database storing (a) a first genetic sequence stringrepresenting a first tissue and (b) a second genetic sequence stringrepresenting a second tissue, wherein the first and second sequencestrings have a plurality of corresponding sub-strings; providing accessto a sequence analysis engine coupled with the genetic database;producing, using the sequence analysis engine, a local alignment byincrementally synchronizing the first and second sequence strings usinga known position of at least one of plurality of correspondingsub-strings; using, by the sequence analysis engine, the local alignmentto generate a local differential string between the first and secondsequence strings within the local alignment; and using, by the sequenceanalysis engine, the local differential string to update a differentialgenetic sequence object in a differential sequence database. In apreferred embodiment, the first and second genetic sequence stringsrepresent at least 10% of a genome, transcriptome, or proteome of thefirst and second tissues, respectively. In an alternative preferredembodiment, the first and second genetic sequence strings represent atleast 50% of a genome, transcriptome, or proteome of the first andsecond tissues, respectively. In another alternatively preferredembodiment, the first and second genetic sequence strings representsubstantially the entire genome, transcriptome, or proteome of the firstand second tissues, respectively. In another preferred embodiment, thecorresponding sub-strings comprise homozygous alleles. In an alternativepreferred embodiment, the corresponding sub-strings compriseheterozygous alleles. In another more preferred embodiment, the geneticsequence object comprises a file. In a yet more preferred embodiment,the file conforms to a standardized format. In a most preferredembodiment, the file conforms to a SAM/BAM format.

In a preferred embodiment, the step of synchronizing comprises aligningat least one of the plurality of sub-strings is based on an a prioriknown location within the first string. In an alternative preferredembodiment the step of synchronizing comprises aligning at least one ofthe plurality of sub-strings based on a known reference stringcomprising known locations for the at least one of the plurality ofsub-strings. In a more preferred embodiment, the known reference stringis a consensus sequence.

In another preferred embodiment, the step of synchronizing comprisesaligning the at least one of the plurality of sub-strings within awindow having a length of less than a length of the at least one of theplurality of sub-strings.

In another preferred embodiment, the differential genetic sequenceobject represents a plurality of local differential strings for at leastone chromosome.

In another preferred embodiment, the differential genetic sequenceobject represents a plurality of local differential strings forsubstantially the entire genome of the first tissue.

In a yet other preferred embodiment, the differential genetic sequenceobject comprises an attribute comprising metadata describing thedifferential genetic sequence object. In a more preferred embodiment,the attribute comprises a state of at least one of the first and secondtissues. In a yet more preferred embodiment, the state comprises aphysiological state of at least one of the first and second tissues. Ina most preferred embodiment, the physiological state comprises a stateselected from the group consisting of neoplastic growth, apoptosis,state of differentiation, tissue age, and responsiveness to treatment.

In an alternative more preferred embodiment, the state comprises geneticstatus. In a most preferred embodiment, the genetic status comprises astatus selected from the group consisting of at least one ploidy, genecopy number, repeat copy number, inversion, deletion, insertion of viralgenes, somatic mutation, germline mutation, structural rearrangement,transposition, and loss of heterozygosity.

In an alternative more preferred embodiment, the state comprises pathwaymodel information associated with a signaling pathway within thetissues. In a most preferred embodiment, the signaling pathway isselected from the group consisting of a growth factor signaling pathway,a transcription factor signaling pathway, an apoptosis pathway, a cellcycle pathway, and a hormone response pathway.

In an alternative embodiment, the first and second tissues originatefrom the same biological entity, the biological entity selected from thegroup consisting of a patient, a healthy individual, a cell line, a stemcell, an experimental animal model, a recombinant bacterial cell, and avirus. In an alternative embodiment, the first tissue is a healthytissue and wherein the second is a diseased tissue. In a more preferredembodiment, the diseased tissue comprises a tumor tissue.

The invention also provides the method as disclosed herein, wherein themethod further comprises the step of iteratively incrementallysynchronizing the first and second sequence strings throughout theentire length of the first sequence string.

The invention also provides a method of providing a health care service,the method comprising: providing access to an analysis engine that isinformationally coupled to a medical records storage device, wherein thestorage device stores a differential genetic sequence object for apatient; producing, by the analysis engine, a patient-specific data setusing presence of a local differential string or constellation of aplurality of local differential strings in the differential geneticsequence object for the patient; and producing, by the analysis engine,a patient-specific instruction based on the patient-specific data set.In a preferred embodiment the medical records storage device isconfigured as a smart-card and is carried by the patient. In anotherpreferred embodiment, the medical records storage device is remotelyaccessible by a healthcare provider. In a yet other preferredembodiment, the differential genetic sequence object for the patientcomprises a plurality of local differential strings for at least twochromosomes. In a still further preferred embodiment, the differentialgenetic sequence object for the patient comprises a plurality of localdifferential strings for substantially the entire genome of the patient.In another preferred embodiment, the differential genetic sequenceobject for the patient comprises a plurality of local differentialstrings representing at least two tissue types, or at least twotemporally spaced results for the same tissue. In a more preferredembodiment, the at least two temporally spaced results for the sametissue are obtained from before and after commencement of a treatment.In a most preferred embodiment, the at least two temporally spacedresults for the same tissue are obtained from before and aftercommencement of a treatment.

In another alternative preferred embodiment, the patient-specificinstruction as disclosed herein is selected from the group consisting ofa diagnosis, a prognosis, a prediction of treatment outcome, arecommendation for a treatment strategy, and a prescription.

The invention also provides a method of analyzing a population, themethod comprising: obtaining and storing a plurality of differentialgenetic sequence objects in a medical records database of a population,wherein the records database is informationally coupled to an analysisengine; identifying, by the analysis engine, a constellation of aplurality of local differential strings within the plurality ofdifferential genetic sequence objects to produce a constellation record;and using, by the analysis engine, the constellation record to generatea population analysis record. In a preferred embodiment, the populationcomprises a plurality of blood relatives. In an alternative preferredembodiment, the population comprises a plurality of memberscharacterized by sharing at least one common feature selected from thegroup consisting of exposure to a pathogen, exposure to a noxious agent,health history, treatment history, treatment success, gender, species,and age. In another alternatively preferred embodiment, the populationcomprises a plurality of members characterized by sharing at least onecommon feature selected from the group consisting of geographiclocation, ethnicity, and occupation. In a still further alternativelypreferred embodiment, the population analysis record comprises paternityor maternity confirmation.

In an alternative embodiment the method disclosed herein furthercomprises a step of comparing a constellation record of an individualpatient with the population analysis record. In a preferred embodiment,the step of comparing of the constellation record of the individualpatient with the population analysis record creates a patient-specificrecord. In a more preferred embodiment, the patient-specific recordcomprises a risk assessment or an identification of the patient asbelonging to a specified population. In an alternative more preferredembodiment, the patient-specific record comprises a diagnosis, aprognosis, a prediction of treatment outcome, a recommendation for atreatment strategy, and a prescription.

The invention further provides a method of analyzing a differentialgenetic sequence object of a person, the method comprising: storing areference differential genetic sequence object in a medical recordsdatabase that is informationally coupled to an analysis engine;calculating, by the analysis engine, a deviation between a plurality oflocal differential strings in the differential genetic sequence objectof the person and a plurality of local differential strings in thereference differential genetic sequence object to produce a deviationrecord; using, by the analysis engine, the deviation record to generatea person-specific deviation profile. In a preferred embodiment, thereference differential genetic sequence object is calculated from aplurality of local differential strings of the person. In anotherpreferred embodiment, the reference differential genetic sequence objectis calculated from a plurality of local differential strings of theperson.

With respect to the various methods disclosed herein, in a preferredembodiment the patient or person is selected from the group consistingof a patient or person diagnosed with a condition, the conditionselected from the group consisting of a disease and a disorder. In amore preferred embodiment, the condition is selected from the groupconsisting of acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, benign prostatic hyperplasia,bronchitis, Chediak-Higashi syndrome, cholecystitis, Crohn's disease,atopic dermatitis, dermnatomyositis, diabetes mellitus, emphysema,erythroblastosis fetalis, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, chronic granulomatousdiseases, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polycystic ovary syndrome, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, severe combinedimmunodeficiency disease (SCID), Sjogren's syndrome, systemicanaphylaxis, systemic lupus erythematosus, systemic sclerosis,thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,complications of cancer, hemodialysis, and extracorporeal circulation,viral, bacterial, fungal, parasitic, protozoal, and helminthicinfection; and adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus, akathesia, Alzheimer'sdisease, amnesia, amyotrophic lateral sclerosis (ALS), ataxias, bipolardisorder, catatonia, cerebral palsy, cerebrovascular diseaseCreutzfeldt-Jakob disease, dementia, depression, Down's syndrome,tardive dyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, muscular dystrophy, neuralgias, neurofibromatosis,neuropathies, Parkinson's disease, Pick's disease, retinitis pigmentosa,schizophrenia, seasonal affective disorder, senile dementia, stroke,Tourette's syndrome and cancers including adenocarcinomas, melanomas,and teratocarcinomas, particularly of the brain.

In another preferred embodiment, the condition is selected from thegroup consisting of cancers such as adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus; immunedisorders such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, trauma, X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; and developmental disorders such as renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, for example, the brain, adrenal gland, kidney, skeletal orreproductive system.

In a still further alternative preferred embodiment, the condition isselected from the group consisting of endocrinological disorders such asdisorders associated with hypopituitarism including hypogonadism,Sheehan syndrome, diabetes insipidus, Kallman's disease,Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis,empty sella syndrome, and dwarfism; hyperpituitarism includingacromegaly, giantism, and syndrome of inappropriate antidiuretic hormone(ADH) secretion (SIADH); and disorders associated with hypothyroidismincluding goiter, myxedema, acute thyroiditis associated with bacterialinfection, subacute thyroiditis associated with viral infection,autoimmune thyroiditis (Hashimoto's disease), and cretinism; disordersassociated with hyperthyroidism including thyrotoxicosis and its variousforms, Grave's disease, pretibial myxedema, toxic multinodular goiter,thyroid carcinoma, and Plummer's disease; and disorders associated withhyperparathyroidism including Conn disease (chronic hypercalemia);respiratory disorders such as allergy, asthma, acute and chronicinflammatory lung diseases, ARDS, emphysema, pulmonary congestion andedema, COPD, interstitial lung diseases, and lung cancers; cancer suchas adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immunological disorderssuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma.

The invention further provides a method of deriving a differentialgenetic sequence object, the method comprising: providing access to agenetic database storing (a) a first genetic sequence stringrepresenting a first tissue and (b) a second genetic sequence stringrepresenting a second tissue, wherein the first and second sequencestrings have a plurality of corresponding sub-strings; providing accessto a sequence analysis engine coupled with the genetic database; usingthe sequence analysis engine to produce a local alignment byincrementally synchronizing the first and second sequence strings usinga known position of at least one of plurality of correspondingsub-strings; using, by the sequence analysis engine, the local alignmentto generate a local differential string between the first and secondsequence strings within the local alignment; and using, by the sequenceanalysis engine, the local differential string to create a differentialgenetic sequence object in a differential sequence database, therebyderiving a differential sequence object.

The invention further provides a transformation method for creating adifferential genetic sequence object, the differential genetic sequenceobject representing a clinically-relevant difference between a firstgenetic sequence and a second sequence, the method comprising the stepsof: (i) providing access to a genetic database storing (a) a firstgenetic sequence string representing a first tissue and (b) a secondgenetic sequence string representing a second tissue, wherein the firstand second sequence strings have a plurality of correspondingsub-strings; (ii) providing access to a sequence analysis engine coupledwith the genetic database; (iii) using the sequence analysis engine toproduce a local alignment by incrementally synchronizing the first andsecond sequence strings using a known position of at least one ofplurality of corresponding sub-strings; (iv) using, by the sequenceanalysis engine, the local alignment to generate a local differentialstring between the first and second sequence strings within the localalignment; and (v) using, by the sequence analysis engine, the localdifferential string to create a differential genetic sequence object ina differential sequence database, thereby deriving a differentialsequence object, wherein the differential sequence object providesobjective information to a user.

In a preferred embodiment, the objective information is selected fromthe group consisting of, genetically relevant information, metabolicallyrelevant information, toxicologically relevant information, clinicallyrelevant information, temporally relevant information, geographicallyrelevant information, occupational risk relevant information, lifehistory relevant information, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 illustrates a schematic of “BamBam” data flow.

FIG. 2 illustrates an overview of allele-specific copy numbercalculation.

FIG. 3 illustrates an overview of structural variation calling.

FIG. 4 illustrates an exemplary method to identify the locations in thegenome where the structural rearrangement occurred.

FIG. 5 illustrates an exemplary tumor-specific genome browser.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments disclosed in this document are illustrative andexemplary and are not meant to limit the invention. Other embodimentscan be utilized and structural changes can be made without departingfrom the scope of the claims of the present invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “an allele” includes aplurality of such alelles, and a reference to “a cluster” is a referenceto one or more clusters and equivalents thereof, and so forth.

As used herein, the term “curated” means the relationships between a setof biological molecules and/or non-biological molecules that has beentested, analyzed, and identified according to scientific and/or clinicalprinciples using methods well known in the art, such as molecularbiological, biochemical, physiological, anatomical, genomic,transcriptomic, proteomic, metabolomic, ADME, and bioinformatictechniques, and the like. The relationships may be biochemical such asbiochemical pathways, genetic pathways, metabolic pathways, generegulatory pathways, gene transcription pathways, gene translationpathways, miRNA-regulated pathways, pseudogene-regulated pathways, andthe like.

High-throughput data is providing a comprehensive view of the molecularchanges in cancer tissues. New technologies allow for the simultaneousgenome-wide assay of the state of genome copy number variation, geneexpression, DNA methylation, and epigenetics of tumor samples and cancercell lines.

Studies such as The Cancer Genome Atlas (TCGA), Stand Up To Cancer(SU2C), and many more are planned in the near future for a wide varietyof tumors. Analyses of current data sets find that genetic alterationsbetween patients can differ but often involve common pathways. It istherefore critical to identify relevant pathways involved in cancerprogression and detect how they are altered in different patients.

With the release of multiple fully-sequenced tumor and matched normalgenomes from projects like The Cancer Genome Atlas (TCGA), there isgreat need for tools that can efficiently analyze these enormousdatasets.

To this end, we developed BamBam, a tool that simultaneously analyzeseach genomic position from a patient's tumor and germline genomes usingthe aligned short-read data contained in SAM/BAM-formatted files(SAMtools library; Li H, Handsaker B, Wysoker A, Fennell T, Ruan J,Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project DataProcessing Subgroup. The Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009 Aug. 15; 25(16):2078-9. Epub 2009 Jun. 8). BamBaminterfaces with the SAMtools library to simultaneously analyze apatient's tumor and germline genomes using short-read alignments fromSAM/BAM-formatted files. In the present disclosure the BamBam tool canbe a sequence analysis engine that is used to compare sequences, thesequences comprising strings of information. In one embodiment, thestrings of information comprise biological information, for example, apolynucleotide sequence or a polypetide sequence. In another embodiment,the biological information can comprise expression data, for examplerelative concentration levels of mRNA transcripts or rRNA or tRNA orpeptide or polypeptide or protein. In another embodiment, the biologicalinformation can be relative amounts of protein modification, such as forexample, but not limited to, phosphorylation, sulphation, actylation,methylation, glycosilation, sialation, modification withglycosylphosphatidylinositol, or modification with proteoglycan.

This method of processing enables BamBam to efficiently calculateoverall copy number and infer regions of structural variation (forexample, chromosomal translocations) in both tumor and germline genomes;to efficiently calculate overall and allele-specific copy number; inferregions exhibiting loss of heterozygosity (LOH); and discover bothsomatic and germline sequence variants (for example, point mutations)and structural rearrangements (for example, chromosomal fusions.Furthermore, by comparing the two genome sequences at the same time,BamBam can also immediately distinguish somatic from germline sequencevariants, calculate allele-specific copy number alterations in the tumorgenome, and phase germline haplotypes across chromosomal regions wherethe allelic proportion has shifted in the tumor genome. By bringingtogether all of these analyses into a single tool, researchers can useBamBam to discover many types of genomic alterations that occurredwithin a patient's tumor genome, often to specific gene alleles, thathelp to identify potential drivers of tumorigenesis.

To determine if a variant discovered is somatic (that is, a variantsequence found only in the tumor) or a germline (that is, a variantsequence that is inherited or heritable) variant requires that wecompare the tumor and matched normal genomes in some way. This can bedone sequentially, by summarizing data at every genomic position forboth tumor and germline and then combining the results for analysis.Unfortunately, because whole-genome BAM files are hundreds of gigabytesin their compressed form (1-2 terabytes uncompressed), the intermediateresults that would need to be stored for later analysis will beextremely large and slow to merge and analyze.

To avoid this issue, BamBam reads from two files at the same time,constantly keeping each BAM file in synchrony with the other and pilingup the genomic reads that overlap every common genomic location betweenthe two files. For each pair of pileups, BamBam runs a series ofanalyses listed above before discarding the pileups and moving to thenext common genomic location. By processing these massive BAM files withthis method, the computer's RAM usage is minimal and processing speed islimited primarily by the speed that the filesystem can read the twofiles. This enables BamBam to process massive amounts of data quickly,while being flexible enough to run on a single computer or across acomputer cluster. Another important benefit to processing these fileswith BamBam is that its output is fairly minimal, consisting only of theimportant differences found in each file. This produces what isessentially a whole-genome diff between the patient's tumor and germlinegenomes, requiring much less disk storage than it would take if allgenome information was stored for each file separately.

BamBam is a computationally efficient method for surveying largesequencing datasets to produce a set of high-quality genomic events thatoccur within each tumor relative to its germline. These results providea glimpse into the chromosomal dynamics of tumors, improving ourunderstanding of tumors' final states and the events that led to them.An exemplary scheme of BamBam Data Flow is shown at FIG. 1.

One particular exemplary embodiment of the invention is creation and useof a differential genetic sequence object. As used herein, the objectrepresents a digital object instantiated from the BamBam techniques andreflects a difference between a reference sequence (for example, a firstsequence) and an analysis sequence (for example, a second sequence). Theobject may be considered a choke point on many different markets. Onemight consider the following factors related to use and management ofsuch objects from a market perspective:

-   -   An object can be dynamic and change with respect to a vector of        parameters (for example, time, geographic region, genetic tree,        species, etc.)    -   Objects can be considered to have a “distance” relative to each        other objects or reference sequences. The distance can be        measured according to dimensions of relevance. For example, the        distance can be a deviation from a hypothetical normal or a        drift with respect to time.    -   Objects can be indicative of risk: risk of developing disease,        susceptibility to exposure, risk to work at a location, etc.    -   Objects can be managed for presentation to stakeholders: health        care providers, insurers, patients, etc.        -   Can be presented as a graphical object        -   Can be presented in a statistical format: single person, a            population, a canonical human, etc.    -   A reference sequence can be generated from the objects to form a        normalized sequence. The normalized sequence can be built based        on consensus derived from measured objects.    -   Objects are representative of large sub-genomic or genomic        information rather than single-gene alignments and are        annotated/contain meta data readable by standard software.    -   Objects can have internal patterns or structures which can be        detected: a set of mutations in one spot might correlate to a        second set of mutations in another spot which correlates to a        condition; constellation of difference patterns could be a hot        spot; use multi-variate analysis or other AI techniques to        identify correlations; detect significance of a hot spot (for        example, presence, absence, etc.)    -   Objects related to a single person could be used as a security        key

Updating a differential sequence object: Update includes creating,modifying, changing, deleting, etc.;

-   -   Can be based on a template    -   Can be a de novo object    -   Can be an existing object

In an alternative exemplary embodiment the method can be used to acertain and predict responsiveness of a patient to treatment:anticipated, assumed, predicted, actual, and the like.

In an alternative exemplary embodiment the method can be used to providepatient-specific instructions: prescription, recommendation, prognosis,and the like.

In one embodiment, the method may be used to provide clinicalinformation that can be used in a variety of diagnostic and therapeuticapplications, such as detection of cancer tissue, staging of cancertissue, detection of metastatic tissue, and the like; detection ofneurological disorders, such as, but not limited to, Alzheimer'sdisease, amyotrophic lateral sclerosis (ALS), Parkinson's disease,schizophrenia, epilepsy, and their complications; developmentaldisorders such as DiGeorge Syndrome, autism, autoimmune disorders suchas multiple sclerosis, diabetes, and the like; treatment of aninfection, such as, but not limited to, viral infection, bacterialinfection, fungal infection, leishmania, schistosomiasis, malaria,tape-worm, elephantiasis, infections by nematodes, nematines, and thelike.

In one embodiment, the method may be used to provide clinicalinformation to detect and quantify altered gene structures, genemutations, gene biochemical modifications, including alterations and/ormodifications to messenger RNA (mRNA), ribosomal RNA (rRNA), transferRNA (tRNA), microRNA (miRNA), antisense RNA (asRNA), and the like, for acondition associated with altered expression of a gene or protein.Conditions, diseases or disorders associated with altered expressioninclude acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, benign prostatic hyperplasia,bronchitis, Chediak-Higashi syndrome, cholecystitis, Crohn's disease,atopic dermatitis, dermnatomyositis, diabetes mellitus, emphysema,erythroblastosis fetalis, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, chronic granulomatousdiseases, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polycystic ovary syndrome, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, severe combinedimmunodeficiency disease (SCID), Sjogren's syndrome, systemicanaphylaxis, systemic lupus erythematosus, systemic sclerosis,thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,complications of cancer, hemodialysis, and extracorporeal circulation,viral, bacterial, fungal, parasitic, protozoal, and helminthicinfection; and adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. The diagnostic assay mayuse hybridization or amplification technology to compare gene expressionin a biological sample from a patient to standard samples in order todetect altered gene expression. Qualitative or quantitative methods forthis comparison are well known in the art.

In another embodiment, the method may be used to provide clinicalinformation to detect and quantify altered gene structures, genemutations, gene biochemical modifications, including alterations and/ormodifications to messenger RNA (mRNA), ribosomal RNA (rRNA), transferRNA (tRNA), microRNA (miRNA), antisense RNA (asRNA), and the like, for adisorder associated with altered expression of a gene or protein.Disorders associated with altered expression include akathesia,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis (ALS),ataxias, bipolar disorder, catatonia, cerebral palsy, cerebrovasculardisease Creutzfeldt-Jakob disease, dementia, depression, Down'ssyndrome, tardive dyskinesia, dystonias, epilepsy, Huntington's disease,multiple sclerosis, muscular dystrophy, neuralgias, neurofibromatosis,neuropathies, Parkinson's disease, Pick's disease, retinitis pigmentosa,schizophrenia, seasonal affective disorder, senile dementia, stroke,Tourette's syndrome and cancers including adenocarcinomas, melanomas,and teratocarcinomas, particularly of the brain.

In one embodiment, the method may be used to provide clinicalinformation for a condition associated with altered expression oractivity of the mammalian protein. Examples of such conditions include,but are not limited to, acquired immunodeficiency syndrome (AIDS),Addison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, benign prostatichyperplasia, bronchitis, Chediak-Higashi syndrome, cholecystitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, chronicgranulomatous diseases, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polycystic ovary syndrome,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, severe combined immunodeficiency disease (SCID), Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infection; and adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus. akathesia,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, ataxias,bipolar disorder, catatonia, cerebral palsy, cerebrovascular diseaseCreutzfeldt-Jakob disease, dementia, depression, Down's syndrome,tardive dyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, muscular dystrophy, neuralgias, neurofibromatosis,neuropathies, Parkinson's disease, Pick's disease, retinitis pigmentosa,schizophrenia, seasonal affective disorder, senile dementia, stroke,Tourette's syndrome and cancers including adenocarcinomas, melanomas,and teratocarcinomas, particularly of the brain.

In yet another embodiment, the method may be used to provide clinicalinformation to detect and quantify altered gene structures, genemutations, gene biochemical modifications, including alterations and/ormodifications to messenger RNA (mRNA), ribosomal RNA (rRNA), transferRNA (tRNA), microRNA (miRNA), antisense RNA (asRNA), and the like, for adisorder associated with altered expression of a gene or protein.Examples of such disorders include, but are not limited to, cancers suchas adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; immune disorders such asacquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, trauma, X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; and developmental disorders such as renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, for example, the brain, adrenal gland, kidney, skeletal orreproductive system.

In another embodiment, the method may be used to provide clinicalinformation to detect and quantify altered gene structures, genemutations, gene biochemical modifications, including alterations and/ormodifications to messenger RNA (mRNA), ribosomal RNA (rRNA), transferRNA (tRNA), microRNA (miRNA), antisense RNA (asRNA), and the like, for adisorder associated with altered expression of a gene or protein.Examples of such a disorder include, but are not limited to,endocrinological disorders such as disorders associated withhypopituitarism including hypogonadism, Sheehan syndrome, diabetesinsipidus, Kallman's disease, Hand-Schuller-Christian disease,Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism;hyperpituitarism including acromegaly, giantism, and syndrome ofinappropriate antidiuretic hormone (ADH) secretion (SIADH); anddisorders associated with hypothyroidism including goiter, myxedema,acute thyroiditis associated with bacterial infection, subacutethyroiditis associated with viral infection, autoimmune thyroiditis(Hashimoto's disease), and cretinism; disorders associated withhyperthyroidism including thyrotoxicosis and its various forms, Grave'sdisease, pretibial myxedema, toxic multinodular goiter, thyroidcarcinoma, and Plummer's disease; and disorders associated withhyperparathyroidism including Conn disease (chronic hypercalemia);respiratory disorders such as allergy, asthma, acute and chronicinflammatory lung diseases, ARDS, emphysema, pulmonary congestion andedema, COPD, interstitial lung diseases, and lung cancers; cancer suchas adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immunological disorderssuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma. Thepolynucleotide sequences may be used in Southern or Northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered nucleic acid sequenceexpression. Such qualitative or quantitative methods are well known inthe art.

Characterization and Best Mode of the Invention

“BamBam” is a computationally efficient method for surveying largesequencing datasets to produce a set of high-quality genomic events thatoccur within each tumor relative to its germline. These results providea glimpse into the chromosomal dynamics of tumors, improving ourunderstanding of tumors' final states and the events that led to them.

Diagnostics

The methods herein described may be used to detect and quantify alteredgene structures, gene mutations, gene biochemical modifications,including alterations and/or modifications to messenger RNA (mRNA),ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), antisenseRNA (asRNA), and the like, for a condition, disease, or disorderassociated with altered expression of a gene or protein, The methodsherein described may be also used to detect and quantify altered geneexpression, absence/presence versus excess, expression of mRNAs or tomonitor mRNA levels during therapeutic intervention. Conditions,diseases or disorders associated with altered expression includeidiopathic pulmonary arterial hypertension, secondary pulmonaryhypertension, a cell proliferative disorder, particularly anaplasticoligodendroglioma, astrocytoma, oligoastrocytoma, glioblastoma,meningioma, ganglioneuroma, neuronal neoplasm, multiple sclerosis,Huntington's disease, breast adenocarcinoma, prostate adenocarcinoma,stomach adenocarcinoma, metastasizing neuroendocrine carcinoma,nonproliferative fibrocystic and proliferative fibrocystic breastdisease, gallbladder cholecystitis and cholelithiasis, osteoarthritis,and rheumatoid arthritis; acquired immunodeficiency syndrome (AIDS),Addison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, benign prostatichyperplasia, bronchitis, Chediak-Higashi syndrome, cholecystitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, chronicgranulomatous diseases, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polycystic ovary syndrome,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, severe combined immunodeficiency disease (SCID), Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, hemodialysis, extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infection; a disorder ofprolactin production, infertility, including tubal disease, ovulatorydefects, and endometriosis, a disruption of the estrous cycle, adisruption of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, an endometrial or ovarian tumor, a uterinefibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis;cancer of the breast, fibrocystic breast disease, and galactorrhea; adisruption of spermatogenesis, abnormal sperm physiology, benignprostatic hyperplasia, prostatitis, Peyronie's disease, impotence,gynecomastia; actinic keratosis, arteriosclerosis, bursitis, cirrhosis,hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,paroxysmal nocturnal hemoglobinuria, polycythemia vera, primarythrombocythemia, complications of cancer, cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In another aspect, thenucleic acid of the invention.

The methods described herein may be used to detect and quantify alteredgene structures, gene mutations, gene biochemical modifications,including alterations and/or modifications to messenger RNA (mRNA),ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), antisenseRNA (asRNA), and the like, for a disorder associated with alteredexpression of a gene or protein. The methods described herein may bealso used to detect and quantify altered gene expression; absence,presence, or excess expression of mRNAs; or to monitor mRNA levelsduring therapeutic intervention Disorders associated with alteredexpression include akathesia, Alzheimer's disease, amnesia, amyotrophiclateral sclerosis, ataxias, bipolar disorder, catatonia, cerebral palsy,cerebrovascular disease Creutzfeldt-Jakob disease, dementia, depression,Down's syndrome, tardive dyskinesia, dystonias, epilepsy, Huntington'sdisease, multiple sclerosis, muscular dystrophy, neuralgias,neurofibromatosis, neuropathies, Parkinson's disease, Pick's disease,retinitis pigmentosa, schizophrenia, seasonal affective disorder, seniledementia, stroke, Tourette's syndrome and cancers includingadenocarcinomas, melanomas, and teratocarcinomas, particularly of thebrain.

In order to provide a basis for the diagnosis of a condition, disease ordisorder associated with gene expression, a normal or standardexpression profile is established. This may be accomplished by combininga biological sample taken from normal subjects, either animal or human,with a probe under conditions for hybridization or amplification.Standard hybridization may be quantified by comparing the valuesobtained using normal subjects with values from an experiment in which aknown amount of a substantially purified target sequence is used.Standard values obtained in this manner may be compared with valuesobtained from samples from patients who are symptomatic for a particularcondition, disease, or disorder. Deviation from standard values towardthose associated with a particular condition is used to diagnose thatcondition.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies and in clinical trial orto monitor the treatment of an individual patient. Once the presence ofa condition is established and a treatment protocol is initiated,diagnostic assays may be repeated on a regular basis to determine if thelevel of expression in the patient begins to approximate the level thatis observed in a normal subject. The assays may also be used to detect,quamtify, or measure gene structures, gene mutations, gene biochemicalmodifications, including alterations and/or modifications to messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA),antisense RNA (asRNA), and the like, that indicate and/or identify thepresence of a tumor, absence of a tumor, or remission status of theindividual undergoing a clinical treatment or therapy. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

The methods disclosed herein may also be used to detect, quantify, andcorrelate a change in gene structures, gene mutations, gene biochemicalmodifications, including alterations and/or modifications to messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA),antisense RNA (asRNA), and the like, that has not been previouslyidentified or associated with a particular clinical disease, disorder,or condition. In the alternative, the methods disclosed herein may beused to identify a novel clinical disease, disorder, or condition. Novelchanges in gene structures, gene mutations, and gene biochemicalmodifications, may then be compared with known chemical and biochemicalproperties of a nucleic acid sequence or protein sequence and whichcorrelate with a clinical disease, disorder, or condition may be used togenerate new databases and knowledge about cellular metabolism forclinical use.

Model Systems

Animal models may be used as bioassays where they exhibit a toxicresponse similar to that of humans and where exposure conditions arerelevant to human exposures. Mammals are the most common models, andmost toxicity studies are performed on rodents such as rats or micebecause of low cost, availability, and abundant reference toxicology.Inbred rodent strains provide a convenient model for investigation ofthe physiological consequences of under- or over-expression of genes ofinterest and for the development of methods for diagnosis and treatmentof diseases. A mammal inbred to over-express a particular gene (forexample, secreted in milk) may also serve as a convenient source of theprotein expressed by that gene.

Toxicology

Toxicology is the study of the effects of agents on living systems. Themajority of toxicity studies are performed on rats or mice to helppredict the effects of these agents on human health. Observation ofqualitative and quantitative changes in physiology, behavior,homeostatic processes, and lethality are used to generate a toxicityprofile and to assess the consequences on human health followingexposure to the agent.

Genetic toxicology identifies and analyzes the ability of an agent toproduce genetic mutations. Genotoxic agents usually have common chemicalor physical properties that facilitate interaction with nucleic acidsand are most harmful when chromosomal aberrations are passed along toprogeny. Toxicological studies may identify agents that increase thefrequency of structural or functional abnormalities in progeny ifadministered to either parent before conception, to the mother duringpregnancy, or to the developing organism. Mice and rats are mostfrequently used in these tests because of their short reproductive cyclethat produces the number of organisms needed to satisfy statisticalrequirements.

Acute toxicity tests are based on a single administration of the agentto the subject to determine the symptomology or lethality of the agent.Three experiments are conducted: (a) an initial dose-range-findingexperiment, (b) an experiment to narrow the range of effective doses,and (c) a final experiment for establishing the dose-response curve.

Prolonged toxicity tests are based on the repeated administration of theagent. Rats and dog are commonly used in these studies to provide datafrom species in different families. With the exception ofcarcinogenesis, there is considerable evidence that daily administrationof an agent at high-dose concentrations for periods of three to fourmonths will reveal most forms of toxicity in adult animals.

Chronic toxicity tests, with a duration of a year or more, are used todemonstrate either the absence of toxicity or the carcinogenic potentialof an agent. When studies are conducted on rats, a minimum of three testgroups plus one control group are used, and animals are examined andmonitored at the outset and at intervals throughout the experiment.

Transgenic Animal Models

Transgenic rodents which over-express or under-express a gene ofinterest may be inbred and used to model human diseases or to testtherapeutic or toxic agents. (See U.S. Pat. Nos. 4,736,866; 5,175,383;and 5,767,337; incorporated herein by reference.) In some cases, theintroduced gene may be activated at a specific time in a specific tissuetype during fetal development or postnatally. Expression of thetransgene is monitored by analysis of phenotype or tissue-specific mRNAexpression in transgenic animals before, during, and after challengewith experimental drug therapies.

Embryonic Stem Cells

Embryonic stem cells (ES) isolated from rodent embryos retain thepotential to form an embryo. When ES cells are placed inside a carrierembryo, they resume normal development and contribute to all tissues ofthe live-born animal. ES cells are the preferred cells used in thecreation of experimental knockout and knockin rodent strains. Mouse EScells, such as the mouse 129/SvJ cell line, are derived from the earlymouse embryo and are grown under culture conditions well known in theart. Vectors for knockout strains contain a disease gene candidatemodified to include a marker gene that disrupts transcription and/ortranslation in vivo. The vector is introduced into ES cells bytransformation methods such as electroporation, liposome delivery,microinjection, and the like which are well known in the art. Theendogenous rodent gene is replaced by the disrupted disease gene throughhomologous recombination and integration during cell division.Transformed ES cells are identified, and preferably microinjected intomouse cell blastocysts such as those from the C57BL/6 mouse strain. Theblastocysts are surgically transferred to pseudopregnant dams and theresulting chimeric progeny are genotyped and bred to produceheterozygous or homozygous strains.

ES cells are also used to study the differentiation of various celltypes and tissues in vitro, such as neural cells, hematopoieticlineages, and cardiomyocytes (Bain et al. (1995) Dev. Biol. 168:342-357; Wiles and Keller (1991) Development 111: 259-267; and Klug etal. (1996) J. Clin. Invest. 98: 216-224). Recent developmentsdemonstrate that ES cells derived from human blastocysts may also bemanipulated in vitro to differentiate into eight separate cell lineages,including endoderm, mesoderm, and ectodermnal cell types (Thomson (1998)Science 282: 1145-1147).

Knockout Analysis

In gene knockout analysis, a region of a human disease gene candidate isenzymatically modified to include a non-mammalian gene such as theneomycin phosphotransferase gene (neo; see, for example, Capecchi (1989)Science 244: 1288-1292). The inserted coding sequence disruptstranscription and translation of the targeted gene and preventsbiochemical synthesis of the disease candidate protein. The modifiedgene is transformed into cultured embryonic stem cells (describedabove), the transformed cells are injected into rodent blastulae, andthe blastulae are implanted into pseudopregnant dams. Transgenic progenyare crossbred to obtain homozygous inbred lines.

Knockin Analysis

Totipotent ES cells, present in the early stages of embryonicdevelopment, can be used to create knockin humanized animals (pigs) ortransgenic animal models (mice or rats) of human diseases. With knockintechnology, a region of a human gene is injected into animal ES cells,and the human sequence integrates into the animal cell genome byrecombination. Totipotent ES cells that contain the integrated humangene are handled as described above. Inbred animals are studied andtreated to obtain information on the analogous human condition. Thesemethods have been used to model several human diseases. (See, forexample, Lee et al. (1998) Proc. Natl. Acad. Sci. 95: 11371-11376;Baudoin et al. (1998) Genes Dev. 12: 1202-1216; and Zhuang et al. (1998)Mol. Cell Biol. 18: 3340-3349).

Non-Human Primate Model

The field of animal testing deals with data and methodology from basicsciences such as physiology, genetics, chemistry, pharmacology andstatistics. These data are paramount in evaluating the effects oftherapeutic agents on non-human primates as they can be related to humanhealth. Monkeys are used as human surrogates in vaccine and drugevaluations, and their responses are relevant to human exposures undersimilar conditions. Cynomolgus monkeys (Macaca fascicularis, Macacamulata) and common marmosets (Callithrix jacchus) are the most commonnon-human primates (NHPs) used in these investigations. Since great costis associated with developing and maintaining a colony of NHPs, earlyresearch and toxicological studies are usually carried out in rodentmodels. In studies using behavioral measures such as drug addiction,NHPs are the first choice test animal. In addition, NHPs and individualhumans exhibit differential sensitivities to many drugs and toxins andcan be classified as “extensive metabolizers” and “poor metabolizers” ofthese agents.

Exemplary Uses of the Invention

Personalized medicine promises to deliver specific treatment(s) to thosepatients mostly likely to benefit. We have shown that approximately halfof therapeutic compounds are preferentially effective in one or more ofthe clinically-relevant transcriptional or genomic breast cancersubtypes. These findings support the importance of definingresponse-related molecular subtypes in breast cancer treatment. We alsoshow that pathway integration of the transcriptional and genomic data onthe cell lines reveals subnetworks that provide mechanistic explanationsfor the observed subtype specific responses. Comparative analysis ofsubnet activities between cell lines and tumors shows that the majorityof subtype-specific subnetworks are conserved between cell lines andtumors. These analyses support the idea that preclinical screening ofexperimental compounds in a well-characterized cell line panel canidentify candidate response-associated molecular signatures that can beused for sensitivity enrichment in early-phase clinical trials. Wesuggest that this in vitro assessment approach will increase thelikelihood that responsive tumor subtypes will be identified before acompound's clinical development begins, thereby reducing cost,increasing the probability of eventual FDA approval and possiblyavoiding toxicity associated with treating patients unlikely to respond.In this study we have assessed only molecular signatures that definetranscriptional subtypes and selected recurrent genome copy numberabnormalities (CNAs). We anticipate that the power and precision of thisapproach will increase as additional molecular features such as geneticmutation, methylation and alternative splicing, are included in theanalysis. Likewise, increasing the size of the cell line panel willincrease the power to assess less common molecular patterns within thepanel and increase the probability of representing a more complete rangeof the diversity that exists in human breast cancers.

Here, we disclose a new software tool we have called BamBam that enablesa rapid comparison of tumor (somatic) and germline matched sequencingdatasets. The results output by BamBam are varied, producing anexhaustive catalogue of the somatic and germline variants contained byeach patient's samples. This catalogue provides researchers with theability to quickly find important changes that occurred during thetumor's development, but also provide high-quality variants present inthe patient's germline that may indicate predisposition to disease.Further improvements of BamBam will consist of methods that specificallysearch for multiple types of variants occurring in the same genomicregion (for example, one allele of a gene deleted, the other allelecontaining a truncating mutation by breakpoint) that may point todrivers of tumorigenesis. We also plan to extend BamBam's ability toprocessing more than pairs of genomes, as well as provide researcherswith the ability to plug in their own analysis methods into BamBam'spipeline.

In additional embodiments, the polynucleotide nucleic acids may be usedin any molecular biology techniques that have yet to be developed,provided the new techniques rely on properties of nucleic acid moleculesthat are currently known, including, but not limited to, such propertiesas the triplet genetic code and specific base pair interactions.

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand not as limitations.

EXAMPLES Example I Dataset Synchronization via the Reference Genome

All short reads are aligned to the same reference genome, making thereference genome a natural way of organizing sequence data frommultiple, related samples. BamBam takes in two short read sequencingdatasets, one from the tumor and the other a matched normal (“germline”)from the same patient, and the reference genome, and reads thesedatasets such that all sequences in both datasets overlapping the samegenomic position are available to be processed at the same time. This isthe most efficient method for processing such data, while also enablingcomplex analyses that would be difficult or impossible to accomplish ina serialized manner, where each dataset is processed by itself, andresults are only merged afterwards.

Such a method is easily extendible to more than two related sequencingdatasets. For example, if three samples, matched normal, tumor, andrelapse, were sequenced, this method could be used to search for changesspecific to the tumor & the relapse sample, and changes specific only tothe relapse, suggesting the relapse tumor has changed somewhat from theoriginal tumor from which it had presumably derived. Also, one could usethis same method to determine the inherited portions of a child's genomegiven sequenced samples from child, father, and mother.

Example II Somatic and Germline Variant Calling

Because BamBam keeps the sequence data in the pair of files in syncacross the genome, a complex mutation model that requires sequencingdata from both tumor and germline BAM files as well as the humanreference can be implemented easily. This model aims to maximize thejoint probability of both the germline genotype (given the germlinereads and the reference nucleotide) and the genotype of the tumor (giventhe germline genotype, a simple mutation model, an estimate of thefraction of contaminating normal tissue in the tumor sample, and thetumor sequence data).

To find the optimal tumor and germline genotype, we aim to maximize thelikelihood defined by

P(D _(g) , D _(t) , G _(g) , G _(t) |α, r)=P(D _(g) −G _(g))P(G _(g)|r)P(D _(t) |G _(g) , G _(t), α)P(G _(t)|G_(g))   (1)

P(D₁g, D₁t, G₁g, G₁t ┤|α, r)=P(D₁g ┤|G₁g)P(G₁g ┤|r)P(D₁t ┤|G₁g, G₁t,α)P(G₁t ┤|G₁g) (1) where r is the observed reference allele, α thefraction of normal contamination, and the tumor and germline genotypesare defined by Gt=(t₁, t₂) and Gg=(g₁, g₂) where t₁, t₂, g₁, g₂ ϵ{A, T,C, G}. The tumor and germline sequence data are defined as a set ofreads D_(t)={d_(t) ¹, d_(t) ², . . . , d_(t) ^(m)} and D_(g)={d_(g) ¹,d_(g) ², . . . , d_(g) ^(m)}, respectively, with the observed basesd_(t) ^(i), d_(g) ^(i) ϵ {A, T, C, G}. All data used in the model mustexceed user-defined base and mapping quality thresholds.

The probability of the germline alleles given the germline genotype ismodeled as a multinomial over the four nucleotides:

${{P( D_{g} \middle| G_{g} )} = {\frac{n!}{{n_{A}!}{n_{T}!}{n_{G}!}{n_{C}!}}{\prod\limits_{i}^{n}{P( d_{g}^{i} \middle| G_{g} )}}}},$

where n is the total number of germline reads at this position andn_(A), n_(G), n_(C), n_(T) are the reads supporting each observedallele. The base probabilities, P(d_(g) ^(i)|G_(g)), are assumed to beindependent, coming from either of the two parental alleles representedby the genotype G_(g), while also incorporating the approximate baseerror rate of the sequencer. The prior on the germline genotype isconditioned on the reference base as

P(G _(g) |r=α)={μ_(aa), μ_(ab), μ_(bb)},

where μ_(aa) is the probability that the position is homozygousreference, μ_(ab) is heterozygous reference, and μ_(bb) is homozygousnon-reference. At this time, the germline prior does not incorporate anyinformation on known, inherited SNPs.

The probability of the set of tumor reads is again defined asmultinomial

${{P( { D_{t} \middle| G_{t} ,G_{g},\alpha} )} = {\frac{n!}{{n_{A}!}{n_{T}!}{n_{G}!}{n_{C}!}}{\prod\limits_{i}^{n}{P( { d_{t}^{i} \middle| G_{t} ,G_{g},\alpha} )}}}},$

where m is the total number of germline reads at this position andm_(A), m_(G), m_(C), m_(T) are the reads supporting each observed allelein the tumor dataset, and the probability of each tumor read is amixture of base probabilities derived from both tumor and germlinegenotypes that is controlled by the fraction of normal contamination, α,as

P(d _(t) ^(i) |G _(t) , G _(g), α)=αP(d _(t) ^(i) |G _(t))+(1−α)P(d _(t)^(i) |G _(g))

and the probability of the tumor genotype is defined by a simplemutation model from on the germline genotype

P(G _(t) |G _(g))=max[P(t ₁ |g ₁)P(t ₂ |g ₂), P(t ₁ |g ₂)P(t ₂ |g ₁)],

where the probability of no mutation (for example, t₁=g₁) is maximal andthe probability of transitions (that is, A→G,T→C) are four times morelikely than transversions (that is, A→T,T→G). All model parameters, α,μ_(aa), μ_(ab), μ_(bb), and base probabilities, P(d^(i)|G) for themultinomial distributions are user-definable.

The tumor and germline genotypes, G_(t) ^(max□), G_(g) ^(max□), selectedare those that maximize (1), and the posterior probability defined by

$\frac{P( {D_{g},D_{t},G_{g}^{{ma}\; x}, G_{t}^{{ma}\; x} \middle| \alpha ,r} )}{\sum\limits_{i,j}^{\;}{P( {D_{g},D_{t},{G_{g} = i},{G_{t} =  j \middle| \alpha },r} )}}$

can be used to score the confidence in the pair of inferred genotypes.If the tumor and germline genotypes differ, the putative somaticmutation(s) will be reported along with its respective confidence.

Maximizing the joint likelihood of both tumor and germline genotypeshelps to improve the accuracy of both inferred genotypes, especially insituations where one or both sequence datasets have low coverage of aparticular genomic position. Other mutation calling algorithms, such asMAQ and SNVMix, that analyze a single sequencing dataset are more likelyto make mistakes when the non-reference or mutant alleles have lowsupport (Li, H., et al. (2008) Mapping short DNA sequencing reads andcalling variants using mapping quality scores, Genome Research, 11,1851-1858; Goya, R. et al. (2010) SNVMix: predicting single nucleotidevariants from next-generation sequencing of tumors, Bioinformatics, 26,730-736).

In addition to collecting allele support from all reads at a givengenomic position, information on the reads are collected (such as whichstrand, forward or reverse, the read maps to, the position of the allelewithin the read, the average quality of the alleles, etc.) and used toselectively filter out false positive calls. We expect a randomdistribution of strands and allele positions for all of the allelesupporting a variant, and if the distribution is skewed significantlyfrom this random distribution (that is, all variant alleles are foundnear the tail end of a read), then this suggest that the variant call issuspect.

Example III Overall and Allele-Specific Copy Number

Overall somatic copy number is calculated using a dynamic windowingapproach that expands and contracts the window's genomic width accordingto the coverage in either the tumor or germline data. The process isinitialized with a window of zero width. Each unique read from eitherthe tumor or germline sequence data will be tallied into tumor counts,Nt, or germline counts, Ng. The start and stop positions of each readwill define the window's region, expanding as new reads exceed theboundaries of the current window. When either the tumor or germlinecounts exceed a user-defined threshold, the window's size and locationare recorded, as well as the Nt, Ng, and relative coverage Nt. Tailoringthe size of the Ng window according to the local read coverage willcreate large windows in regions of low coverage (for example, repetitiveregions) or small windows in regions exhibiting somatic amplification,thereby increasing the genomic resolution of amplicons and increasingour ability to define the boundaries of the amplification.

Allele-specific copy number is calculated similarly, except that onlypositions deemed heterozygous in the germline are included, as shown(see FIG. 2). Heterozygosity is defined as a position in the germlinethat is believed to have two different alleles, one allele contributedby each parent. Majority and minority copy numbers are calculated usingthe same dynamic windowing technique described above for overall copynumber in order to aggregate data in the same genomic neighborhood. Themajority allele at a heterozygous site is defined herein as the allelethat has the greatest number of supporting reads in the tumor datasetthat overlap that genomic location, while the minority allele is allelethat has the least support. All counts ascribed to the majority allelein both tumor and germline data will go towards calculation of themajority copy number, and similarly for the minority allele. Themajority and minority allele counts are then normalized by the counts ofboth alleles in the germline data, Ng, to calculate majority andminority copy numbers.

Allele-specific copy number is used to identify genomic regionsexhibiting loss-of-heterozygosity (both copy-neutral and copy-loss) aswell as amplifications or deletions specific to a single allele. Thislast point is especially important to help distinguish potentiallydisease-causing alleles as those that are either amplified ornot-deleted in the tumor sequence data. Furthermore, regions thatexperience hemizygous loss (for example, one parental chromosome arm)can be used to directly estimate the amount of normal contaminant in thesequenced tumor sample, which can be used to improve the modeling of thegermline and tumor genotypes described above.

FIG. 2 shows an overview of allele-specific copy number calculation.Positions with heterozygous genotypes are determined using both germlineand tumor sequencing data, as determined by the germline variant callingalgorithm. All reads overlapping these locations are collected and theread support for each of the two alleles in the heterozygous genotypeare found in both tumor and germline. The majority allele is determinedto be the allele with the highest support, and majority copy number iscalculated by normalizing this count by the overall number of reads atthat position in the germline.

Example IV Phasing Genotypes

BamBam attempts to phase all heterozygous positions found in thegermline by taking advantage of allelic imbalance caused by large scalegenomic amplifications or deletions in the tumor. The majority vote basecall is selected at every position in the tumor sequence data toconstruct the phased haplotype present in the tumor. The majority votechooses the most abundant allele observed in the pool of short reads,which should select the allele that remains in the tumor after adeletion event or the duplicated allele of an amplification event. Ateach position, the allelic state of the germline is also identified,where a position is deemed homozygous if there exists only one allelewith the requisite read support and heterozygous if at least two alleleshave the required read support. The tumor's haplotype is assumed torepresent one of the two parental haplotypes, where the second parentalhaplotype is derived as the sequence of germline alleles that do notbelong to the tumor haplotype. This procedure is used genome-wideregardless of the allelic proportion in the tumor, so we expect thehaplotype assignment of genotypes to be essentially random in regionsthat are equally balanced between major and minor alleles. Accuratephasing of germline sequence will only occur in regions that exhibit aconsistent allelic imbalance resulting from a single genomic event (forexample regional amplification or deletion) in the tumor. Validation ofthe tumor-derived haplotypes can be accomplished by comparing thetumor-derived haplotypes to phased genotypes available from the HapMapproject (International HapMap Consortium (2007), Nature, 7164: 851-861).

Example V Inferring Structural Variation Using Paired-End Clustering

To identify putative intra- and inter-chromosomal rearrangements, BamBamsearches for discordant paired reads where each read in the pair map todisparate regions of the reference sequence. Intra-chromosomaldiscordant pairs are those that have an abnormally large insert size(i.e. the genomic distance on the reference separating the paired readsexceeds a user-defined threshold) or those that map in an incorrectorientation (i.e. inversion). Inter-chromosomal discordant pairs aredefined by paired reads that map to different chromosomes. Alldiscordant paired-end reads that align to identical locations as otherpairs are removed to avoid calling rearrangements supported by a largenumber of reads that are merely the result of the PCR amplification stepin the short-read library's preparation. An overview of this process isshown in FIG. 3.

All discordant paired-end reads are clustered according to their genomiclocations to define an approximate genomic region where the breakpointis believed to be. The aggregation process consists of grouping togetherthe unique reads that overlap other reads on both sides of the putativebreakpoint. The strand orientation of all overlapping reads must alsomatch or are not include in the cluster of pairs. When the number ofoverlapping discordant pairs in a cluster exceeds a user-definedthreshold, the breakpoint that describes the rearrangement is defined.If there are rearrangements present in both germline and tumor datasetsat the same position, then they are compared as follows. Germlinerearrangements require that the tumor and germline dataset support thesame rearrangement since it is exceedingly unlikely that a structuralvariation observed in the germline would somehow be reversed in thetumor to precisely agree with the reference. On the other hand, somaticrearrangements must only be observed in the tumor sequencing data, andnot substantially present in the germline dataset. Rearrangements thatfulfill these requirements are stored for post-processing analysis andvisualization, while those that do not are discarded as artifactualrearrangements caused by either the sequencing instrument, samplepreparation (such as whole-genome amplification), or a systematic biasof the short-read mapping algorithm employed.

FIG. 3 shows an overview of structural variation calling. The initialidentification of a putative structural variant is identified by BamBamusing discordantly mapped read pairs, where both reads fully map to thereference genome, but do so in an abnormal, non-reference manner. Theputative breakpoints found by BamBam are then refined by a programcalled bridget using any available split-reads.

Example VI Refinement of Structural Variation Using Split-Reads

The breakpoints found initially by BamBam are approximate, in that theyuse fully-mapped reads that, by their nature, cannot overlap the actualjunction of the breakpoint, since it represents sequence not present inthe reference (or the germline dataset, in the case of a somaticrearrangement). To refine our knowledge of the location of thebreakpoint, a program called Bridget was developed, which is summarizedin FIG. 4.

Bridget is given the approximate breakpoint found by BamBam and searchesfor all unaligned reads that are anchored near the putative breakpointby a fully-mapped mate. Each of these unmapped reads have the potentialto be “split reads” that overlaps the rearrangement's breakpointjunction. Localized genomic sequences surrounding both sides of thebreakpoint are broken up into a set of unique tiles (currently tilesize=16 bp), and a tile database of the tile sequences and theirlocation in the reference genome is built. A similar tile database isconstructed for each unaligned read, by breaking up the read into tilesof the same size and noting their location within the read. Comparingthe reference tile database and the unaligned tile database, the genomiclocation of each unaligned tile in the reference is determined. “Dualspanning sets” of these locations are computed by determining themaximal set of tiles that are contiguous in BOTH the reference andunaligned reads, one for each side of the breakpoint.

The minimum and maximum genomic locations of the “dual spanning sets” inreference coordinates precisely determine the breakpoint location, aswell as the orientation (or strandedness) of the sequence. With theinformation describing the left and right boundaries of the breakpoint,the rearranged sequence is fully defined, that is, the left side isdefined by (chromosome=chr1, location=1000 bp, strand=forward) and theright side is defined by (chromosome=chr5, location=500,000 bp,strand=reverse). The sequence homology of the breakpoint (that is, ashort sequence, such as “CA,” observed to be identical on bothboundaries of the breakpoint, but is observed only once in the alignedread at the junction of the two sequences) is also determined from thesedual spanning sets.

For each unaligned read, the dual spanning sets determine a potentiallocation of the breakpoint. Since each unaligned read may determineslightly different locations for the breakpoint (due to sequence errorsnear the breakpoint, repetitive reference, etc.), all breakpointlocations determined from the dual spanning sets are used to generatepossible junction sequences. All unmapped reads are newly aligned toeach of these possible junction sequences and the overall improvement intheir alignments is measured against how well the reads aligned to theoriginal sequences. The junction sequence that yields the greatestimprovement in alignment scores is judged as the best candidate for thetrue rearrangement. If this best junction sequence yields little-to-noimprovement in the alignment scores, then this junction sequence isdiscarded as it is unlikely to represent the true rearrangement. In thiscase, it may also be determined that the lack of split-read confirmationis evidence that the original structural rearrangement found by BamBamcould be artifactual.

FIG. 4 shows an exemplary method to precisely identify the locations inthe genome where the structural rearrangement occurred. Tiles (or kmers)are determined for both the potential split read and the referencegenome. Dual spanning sets are determined (represent as the thick redand purple boxes on the bottom of this figure), which fully define howto construct the rearranged sequence. Dual spanning sets are robust tosequence errors or SNPs in the split read.

Example VII Tumor-Specific Genome Browser

To visualize all of the results output by BamBam, a tumor genome browserwas developed that simultaneously displays all of the genomic variantsfound in a single tumor sample, versus its matched normal, as shown inFIG. 5. It is capable of displaying overall & allele specific copynumber, intra- and inter-chromosomal rearrangements, and mutations andsmall indels. It displays data in both linear and circular plots, thelatter of which being much better suited for display inter-chromosomalrearrangements.

By displaying the data together in a single image, the user can quicklynavigate a single sample's data and understand the relationship betweenchanges in copy number and a structural variation. For example, a largeintra-chromosomal deletion-type rearrangement should have a concordantdrop in copy number in the region between the breakpoints. Also,displaying mutation data with copy number data allows the user tounderstand if a somatic mutation was subsequently amplified, or if thewild-type allele was deleted in the tumor, both vital datapointssuggesting the importance of the genomic locus in this sample'stumorigenesis.

FIG. 5 shows an exemplary tumor-specific genome browser. The browsershows all of the high-level somatic difference discovered by BamBam in asingle image, enabling the synthesis of multiple distinct datasets togive an overall picture of the tumor's genome. The browser is able tozoom into and out of genomic regions rapidly, going from the full genomeview, as shown above, to a single base resolution in just a few clicks.

Example VIII Computational Requirements

Both BamBam and Bridget were written in C, requiring only standard Clibraries and the latest SAMtools source code (available fromhttp://samtools.sourceforge.net). It may be run as a single process orbroken up into a series of jobs across a cluster (for example, one jobper chromosome). Processing a pair of 250 GB BAM files, each containingbillions of 100 bp reads, BamBam will finish its whole-genome analysisin approximately 5 hours as a single process, or about 30 minutes on amodest cluster (24 nodes). BamBam's computational requirements werenegligible, requiring only enough RAM to store the read data overlappinga single genomic position and enough disk space to store thewell-supported variants found in either tumor or germline genomes.

Bridget also had very modest computational requirements. Runtimes on asingle machine were typically less than a second, which includes thetime necessary to gather the reference sequence and any potentialsplit-reads in the neighborhood of a breakpoint, build tile databasesfor both reference and split-reads, determine all dual spanning sets,construct potential junction sequences, re-align all split-reads to bothreference and each junction sequence, and determine the best junctionsequence. Regions that are highly amplified or have high numbers ofunmapped reads increase the running time of Bridget, but this may bemitigated by the easy parallelizability of Bridget.

Example IX Isolation of Genomic DNA

Blood or other tissue samples (2-3 ml) are collected from patients andstored in EDTA-containing tubes at −80° C. until use. Genomic DNA isextracted from the blood samples using a DNA isolation kit according tothe manufacturer's instruction (PUREGENE, Gentra Systems, MinneapolisMinn.). DNA purity is measured as the ratio of the absorbance at 260 and280 nm (1 cm lightpath; A₂₆₀/A₂₈₀) measured with a Beckmanspectrophotometer.

Example X Identification of SNPs

A region of a gene from a patient's DNA sample is amplified by PCR usingthe primers specifically designed for the region. The PCR products aresequenced using methods well known to those of skill in the art, asdisclosed above. SNPs identified in the sequence traces are verifiedusing Phred/Phrap/Consed software and compared with known SNPs depositedin the NCBI SNP databank.

Example XI Statistical Analysis

Values are expressed as mean±SD. χ² analysis (Web Chi Square Calculator,Georgetown Linguistics, Georgetown University, Washington DC) is used toassess differences between genotype frequencies in normal subjects andpatients with a disorder. One-way ANOVA with post-hoc analysis isperformed as indicated to compare hemodynamics between different patientgroups.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described embodiments can be configuredwithout departing from the scope and spirit of the invention. Othersuitable techniques and methods known in the art can be applied innumerous specific modalities by one skilled in the art and in light ofthe description of the present invention described herein. Therefore, itis to be understood that the invention can be practiced other than asspecifically described herein. The above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

What is claimed:
 1. A sequence analysis computer system comprising: afile system storing a first digital sequence file and a different,second digital sequence file wherein each file includes sequence shortreads that are aligned with respect to sequence locations; and at leastone computer operating as a sequence analysis engine that is configuredto: identify in the first and second digital sequence files a firstcommon sequence location from the sequence locations; read from thefirst digital sequence file a first set of sequence short reads thatoverlap with the first common sequence location, thereby forming a firstpileup stored in a memory; read from the second digital sequence file asecond set of sequence short reads that overlap with the first commonsequence location, thereby forming a second pileup stored in the memory;identify at least one variant as a difference among inferred bases atthe first common sequence location as a function of the short reads inthe first pileup and the short reads in the second pile that overlap thecommon sequence location by optimizing likelihoods of the inferred basesbased on observed bases of the first and second piles at the commonsequence location; and output the at least one variant in a file as asequence difference between the first digital sequence file and thesecond digital sequence file.
 2. The system of claim 1, wherein the atleast one computer comprises a cluster of networked computers operatingas the sequence analysis engine.
 3. The system of claim 1, wherein thefirst set of sequence short reads and the second set of sequence shortreads are read from the their respective files at the same time.
 4. Thesystem of claim 3, wherein the sequence analysis engine is furtherconfigured to synchronize reading short reads with respect to thesequence locations from the first and the second digital sequence files.5. The system of claim 1, wherein only short reads in the first pileupand second pile that overlap the common sequence location are read intothe memory.
 6. The system of claim 1, wherein the first sequence filecomprises a SAM formatted file or a BAM formatted file.
 7. The system ofclaim 1, wherein the second sequence file comprises a SAM formatted fileor a BAM formatted file.
 8. The system of claim 1, wherein the sequenceanalysis engine is further configured to move to a next common sequencelocation after analyzing the first and second pileups as the firstcommon sequence location.
 9. The system of claim 8, wherein the sequenceanalysis engine is further configured to discard short reads from thefirst pileup and the second pileup that do not overlap with the nextcommon sequence location.
 10. The system of claim 1, wherein the firstand the second digital sequence files both comprise genomic sequences.11. The system of claim 10, wherein the first digital sequence filecomprises sequences from a normal genome of a normal tissue sample. 12.The system of claim 10, wherein the second file comprise sequences froma diseased genome of a diseased tissue sample.
 13. The system of claim12, wherein the diseased genome represents a tumor genome from a tumortissue.
 14. The system of claim 10, wherein the first digital sequencefile comprises tumor sequences from a tumor genome of a tumor tissue andthe second digital sequence file comprises normal sequences from amatched-normal tissue.
 15. The system of claim 10, wherein the at leastone variant comprises a somatic variant.
 16. The system of claim 10,wherein the at least one variant comprises a germline variant.
 17. Thesystem of claim 1, wherein the at least one variant comprises at leastone of the following: a structural variation, a point mutation, achromosomal fusion, a chromosomal translocation, a loss ofheterozygosity, at least one ploidy, an inversion, a deletion, aninsertion, a somatic mutation, a germline mutation, and a transposition.18. The system of claim 1, wherein the at least one variant stored assequence difference includes metadata related to the difference.
 19. Thesystem of claim 18, wherein the metadata includes at least one of thefollowing attributes: a tissue state, a genetic state, a physiologicalstate, and a signaling pathway.
 20. The system of claim 1, wherein thesequence analysis engine is further configured to filter the at leastone variant as a false positive as a function of one or more pieces ofinformation on the short reads including at least one of the following:a mapping to a forward strand, a mapping to a reverse strand, a positionof an allele in a read, and an average quality of a read.
 21. The systemof claim 1, wherein the sequence locations comprise locations of areference sequence.
 22. The system of claim 1, wherein the sequencelocations are obtained from the first and second digital sequence files.